Chronic ethanol consumption has profound effects on signal transduction. Our long-term objectives are to understand the effects of chronic ethanol exposure on signal transduction in neuronal systems in order to elucidate the mechanisms of ethanol toxicity and potential therapies for alcoholism. Recent data, using PC12 cells as a model, suggests that long-term ethanol has effects on specific signal transduction systems. In cells exposed to ethanol chronically, stimulation of phospholipase D by bradykinin (BK) and other agents is greatly attenuated, whereas activation of phospholipase C by BK is not altered. Thus, these two pathways adapt differently to the presence of ethanol. The attenuation of activation of phospholipase D represents a new and potentially important effect of long-term ethanol exposure, since this pathway leads to the production of second messengers such as phosphatidic acid and diacylglycerol. The above data suggest that there are differences in the way BK activates these two phospholipases. Phosphorylation is a critical event in the activation of phospholipase D, while phospholipase C appears to be regulated by GTP binding proteins. Preliminary data demonstrate that all the agents that activate phospholipase D, also cause phosphorylation of the enzyme on serine residues. Inhibition of serine phosphatase enhances BK stimulated phospholipase D. Long-term ethanol and higher concentrations of okadaic acid enhance phosphorylation of phospholipase D and attenuate activitation. Thus. phosphorylation of some serine residues facilitates activation while phosphorylation of additional residues leads to inhibition. We propose to characterize the role of serine phosphorylation in the regulation of phospholipase D and thereby learn the role of long- term ethanol exposure in the attenuation of activation. Aim 1) Characterize the role of phosphorylation in the activation/inhibition of phospholipase D. Aim 2) Characterize the sites of phosphorylation on phospholipase D induced by agonists and long-term ethanol. For aims 1 and 2 and we will use cells transfected with phospholipase D2 containing an influenza HA tag. Phospholipase D will be immunoprecipitated with anti-HA antibody. Aim 3) Characterize the role of an ethanol induced activation of protein kinase C in the down regulation of phospholipase D. We will use cell lines transfected with protein kinase C-epsilon, the enzyme that is up regulated during chronic ethanol exposure. All the techniques to perform this work are available in my laboratory. Preliminary data support the hypothesis. Furthermore, this is a very focused set of experiments that should yield reliable data. The results will have important ramifications in understanding the effects of chronic ethanol on specific signal transduction pathways and the regulation of this important enzyme, phospholipase D.